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Biochemistry (Moscow)

, Volume 83, Issue 10, pp 1255–1262 | Cite as

The Expression of Matryoshka Gene Encoding a Homologue of Kunitz Peptidase Inhibitor Is Regulated Both at the Level of Transcription and Translation

  • E. V. Sheshukova
  • T. V. Komarova
  • N. M. Ershova
  • A. M. Bronstein
  • Y. L. DorokhovEmail author
Short Communications
  • 25 Downloads

Abstract

The gene for Kunitz peptidase inhibitor-like protein (KPILP) contains nested alternative open reading frame (aORF) that controls expression of the maternal mRNA. The content of NbKPILP mRNA in intact leaves of Nicotiana benthamiana plant is low but increases significantly upon extended dark exposure or when foreign nucleic acid is overexpressed in the cells. The NbKPILP gene promoter along with the expressed nested aORF are likely to play an important role in maintaining the levels of NbKPILP mRNA. To elucidate the role of NbKPILP promoter, we isolated a fragment of N. benthamiana chromosomal DNA upstream of the NbKPILP transcription start, sequenced it, and created constructs in which reporter E. coli uidA gene coding for β-D-glucuronidase (GUS) was placed under control of the NbKPILP promoter. By assessing the efficacy of uidA mRNA synthesis directed by the NbKPILP promoter and 35S promoter of the cauliflower mosaic virus in a transient expression system, we showed that the levels of GUS accumulation were comparable for both promoters. Prolonged incubation of the agroinjected plants in the darkness stimulated accumulation of the uidA mRNA directed by the NbKPILP promoter. Our experiments indicate that along with regulation at the transcriptional level, expression of NbKPILP mRNA can be affected by expression of the nested aORF controlled by the polypurine block (PPB) located upstream of its start codon, since introduction of mutations in the PPB resulted in significant accumulation of the NbKPILP mRNA. Nucleotide replacement in the aORF start codon led to the drastic increase in the amounts of NbKPILP mRNA and its protein product.

Keywords

Agrobacterium tumefaciens transient expression Kunitz peptidase inhibitor matryoshka gene promoter translation abiotic factor polypurine block 

Abbreviations

aORF

nested alternative reading frame

GUS

β-D-glucuronidase

KPI

Kunitz peptidase inhibitor

(Nb)KPILP

(Nicotiana benthamiana) Kunitz peptidase inhibitor-like protein

PPB

polypurine block

proNbKPILP

NbKPILP promoter

RT-qPCR

quantitative reverse transcription polymerase chain reaction

35S

cauliflower mosaic virus (CaMV) 35S RNA promoter

(s)ORF

(short) ORF

sPEP

peptide encoded by sORF

3′-UTR

3′-untranslated region

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Copyright information

© Pleiades Publishing, Ltd. 2018

Authors and Affiliations

  • E. V. Sheshukova
    • 1
  • T. V. Komarova
    • 1
    • 2
  • N. M. Ershova
    • 1
    • 2
  • A. M. Bronstein
    • 3
  • Y. L. Dorokhov
    • 1
    • 2
    Email author
  1. 1.Vavilov Institute of General GeneticsRussian Academy of SciencesMoscowRussia
  2. 2.Belozersky Institute of Physico-Chemical BiologyLomonosov Moscow State UniversityMoscowRussia
  3. 3.Sechenov First Moscow State Medical UniversityMoscowRussia

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